Previous reports have suggested that the two mitogen-activated protein kinases (MAPKs) in and cells revealed that the phosphorylation of ERK1 could be mediated through an intercellular signal other than folate. and that other cell-cell signaling mechanisms contribute to this activation. G5 subunit signaling can down regulate ERK1 function to promote prestalk cell development but not through major changes to the level of phosphorylated ERK1. has only two MAPKs, ERK1 and ERK2 (39% primary sequence identity), and both play important roles in the developmental life cycle that allows solitary cells to aggregate and develop into a fruiting body structure consisting of a stalk and a mass of spores [3]. During this multicellular development, intercellular signaling mediated through G protein coupled receptors regulates the differentiation and sorting of prespore and prestalk cells within the aggregate and some of these signaling pathways involve MAPK activity. ERK2 function is essential for the cell aggregation process in which cells respond to and produce an extracellular cAMP signal that allows cells to chemotax toward each other [14C16]. The stimulation of cell surface cAMP receptors leads to ERK2 activation but interestingly this activation does not require the G2 and G subunits of the G protein that couples with cAMP receptors [17, 18]. Genetic analysis has indicated that ERK2 down regulates the cAMP-specific phosphodiesterase RegA and thereby allows the accumulation of cAMP for internal signaling and the relay of external cAMP signaling [19]. A loss in ERK2 function results in an aggregation defective developmental phenotype because insufficient cAMP accumulates to relay 108341-18-0 IC50 the cAMP signal to other cells [15]. This aggregation deficiency can be corrected by developing cells in chimeric populations with wild-type cells that produce sufficient extracellular cAMP signaling but cells do not sort properly within these chimeric aggregates [14, 20, 21]. The aggregation deficiency of cells can also be suppressed by disrupting the gene to reduce cAMP phosphodiesterase activity [15, 19]. ERK2 is also activated in response to extracellular folate, another chemoattractant, and this activation does require the G4 and G subunit that couple to folate receptors [22, 23]. use this G4-mediated signaling pathway to forage for new bacterial food sources [24]. The activation and function of ERK1 108341-18-0 IC50 in development has not been characterized as well as the developmental role of ERK2. Earlier reports have suggested that ERK1 can be activated in response to external cAMP signaling and that this activation can be mediated by MEK1, the only MAP2K identified by sequence similarity to other eukaryotic MAP2Ks [25, 26]. Loss of MEK or ERK1 function results in small aggregate formation and accelerated development consistent with both kinases functioning in the same pathway. A major challenge in characterizing ERK1 function has been the inability to detect phosphorylated ERK1 using antibodies that recognize the phosphorylated TXY motif of other ICAM2 MAPKs, such as ERK2 [21]. While 108341-18-0 IC50 the analysis of ERK1 activation has been quite limited, the phenotypic analysis of erk1- mutants suggests that ERK1 plays an important role in determining aggregate size and the rate of development. Developmental signaling pathways mediated by 108341-18-0 IC50 the G5 subunit can also regulate aggregate size and rate of development suggesting a possible connection between G5 and ERK1 function [27]. The phenotypic analyses of and strains indicate the two MAPKs have different roles in development but some studies have suggested these MAPKs are co-activated in response to cAMP. In this report we identified an antibody that can detect the phosphorylated ERK1 protein and demonstrate that the phosphorylation of ERK1 does not occur synchronously with the phosphorylation of ERK2 when cells are stimulated by cAMP. The phosphorylation of ERK1 in response to folate was also examined because MAP2K activity is present to activate ERK2. The phosphorylation of ERK1 or ERK2 was examined in cells deficient in the other MAPK to evaluate whether or not the activation of one MAPK was dependent on the other. Finally, the role of ERK1 function in G5 signaling pathways was examined through an epistasis analysis of and mutations and an analysis 108341-18-0 IC50 of phosphorylated ERK1 in mutants. The results of this study suggest different mechanisms exist for the regulation of ERK1 and ERK2 function and that these differences might reflect the different contributions these MAPKs provide in development. 2. Materials and methods 2.1. Media and Strains All traces were isogenic with the wild-type stress KAx-3 except for the noted mutations. The creation of the traces provides been reported [21 previously, 23, 28, 29]. New traces in KAx-3 and JH10 backdrops had been made, using the previously defined gene interruption build and technique because the preliminary stress do not really display the expanded advancement phenotype defined in various other reviews [25], the result of secondary mutations possibly. The erk1 gene was interrupted in a.

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